Many diseases are caused by aberrant levels of proteins which cause cellular responses to be either heightened or dampened. Under normal conditions, proteins are targeted for proteasomal degradation by the ubiquitination pathway. Ubiquitination is the post-translational modification of a protein by the covalent attachment of one or more ubiquitin monomers.
The cascade leading to ubiquitination by a ubiquitination ligase (E3) is a complex enzymatic process with numerous proteins involved. There are three different catalytic events: ubiquitin activation; ubiquitin conjugation and ubiquitin ligation.
Ubiquitin is first activated in an ATP-dependent manner by a ubiquitin activating enzyme (E1). The C-terminus of a ubiquitin forms a high energy thiolester bond with E1. The ubiquitin is then passed to a ubiquitin conjugating enzyme (E2; also called ubiquitin carrier protein), also linked to this second enzyme via a thiolester bond. The ubiquitin is finally linked to its target protein to form a terminal isopeptide bond under the guidance of a ubiquitin ligase (E3). In this process, chains of ubiquitin are formed on the target protein, each covalently ligated to the next through the activity of E3.
E1 and E2 are structurally related and well characterized enzymes. There are several species of E2 (at least 25 in mammals), some of which act in preferred pairs with specific E3 enzymes to confer specificity for different target proteins.
E3 enzymes contain two separate activities: a ubiquitin ligase activity to conjugate ubiquitin to substrates and form polyubiquitin chains via isopeptide bonds, and a targeting activity to bring the ligase and substrate physically together. Substrate specificity of different E3 enzymes is the major determinant in the selectivity of the ubiquitin-dependent protein degradation process.
As the ubiquitination cascade provides important targets for therapeutics, it is crucial to have an effective in vitro ubiquitination assay. In particular, it is important that such an assay can be used for high-throughput screening of potential ubiquitination cascade modulators.
Ubiquitination assays are known in the art. In U.S. Pat. No. 6,413,725, the level of ubiquitination of a particular substrate is measured by monitoring changes to the molecular weight of the substrate as a marker of ubiquitination activity. The assay is specific for the Cdc53 and Cdc-53 related human cullin E3 ubiquitin ligases and their limited substrates, e.g. Sic1. As the assay relies on changes in molecular weight of the substrate, direct measurement of ubiquitination over the time course of the reaction is difficult and the ubiquitinated substrates are preferably analysed after completion of the reaction, i.e. via SDS PAGE separation. This limitation makes the assay inefficient for high-throughput screening.
The ubiquitination assay described in EP1268847 measures E3 ligase activity by monitoring the amount of poly-ubiquitin bound to the ligase itself. The ligase and ubiquitin are labelled with a FRET pair. As the ligase is labelled with one member of the FRET pair, it is directly involved in detection of ubiquitination. Furthermore, the ubiquitination measured is ubiquitination of the ligase itself. Therefore, the assay is limited to use in detection of auto-ubiquitination of a single E3 ligase.
Accordingly, although assays for ubiquitination already exist in the art, these assays have limited application. There is therefore a need in the art for improved ubiquitination assays which overcome the disadvantages associated with the assays known in the art and can be used for efficient high throughput screening of ubiquitination cascade modulators.